For a number of flaviviruses, in particular for those associated with neuroinvasion and with the encephalitic syndrome such as West Nile, Japanese encephalitis or tick-borne encephalitis viruses, expression of the virulent phenotype at least in part depends on determinants in the envelope protein. In general, all known to date attenuating mutations affect the main functionality of the E protein, which plays the major role at the phase of flavivirus entry by fusion. This application is designed to investigate effects of a previously unknown mutations that influence the strength of monomer interaction in the E dimer existing on the surface of infectious virions. We hypothesize that mutations increasing the strength of monomer association will be less detrimental for virus assembly than for the virus infectivity. Such mutations will have minimal or no effect on virion assembly at the end of the viral infectious cycle, yet may lead to virus attenuation due to interference at the initial phase of the next reproductive cycle. To test this hypothesis, the following specific aims have been defined in this pilot study: 1) To investigate effect of mutations at the identified contact interface on formation and the stability of the protein E dimers. We will analyze biochemical properties, such as dimerization, the dimer stability and dissociation kinetics of mutagenized E400 ectodomains produced in the Drosophila expression system. Mutations that are expected to influence the dimer stability have been selected by homology modeling of the NY99 E400 ectodomain using available coordinates of the DEN2 and DENS E dimers in the profusion conformation. 2) To evaluate effect of mutations at the monomer contact interface on the biological properties of WN virus. E proteins carrying the selected mutations will be inserted into the NY99 infectious DNA construct and biological properties of recovered viruses, such as growth characteristics in tissue culture and virulence in mice, will be investigated. Formation of the E dimer is crucial both for virion assembly and for the realization of its infectious properties. The goal of this pilot study is to determine if selective mutagenesis at the contact interface can influence dimer formation and its stability and whether the observed differences will lead to differential effects on virus biological properties. Results of the proposed study will indicate if manipulation of the strength of monomer association in the E dimer found in the envelope of infectious flavivirus virions can offer new means of virus attenuation. [unreadable] [unreadable] [unreadable]